Lung cancer is the most common cancer and the leading cause of cancer-related deaths worldwide (Ezzati M, et al. Lancet 2003; 362:847-52). More than 1.4 million people die of lung cancer each year (Jemal A, et al. CA Cancer J Clin 2008; 58:71-96.). Lung cancer is also the first frequently diagnosed cancer in Taiwan, and it accounts for 20% cancer deaths (Sinchaikul S, et al. Chang Gung Med J 2008; 31:417-30.). In the United States, it is estimated that 219,440 men and women (116,090 men and 103,350 women) were diagnosed with, and 159,390 men and women died of, cancer of the lung and bronchus in 2009 (Surveillance Research Program, National Cancer Institute. seer.cancer.gov/faststats; accessed on Feb. 1, 2010)
Lung cancers, also known as bronchogenic carcinomas, are broadly classified clinically into two types: small cell lung cancers (SCLC) and non-small cell lung cancers (NSCLC). This classification is based upon the microscopic appearance of the tumor cells themselves. These two types of cancers grow and spread in different ways and may have different treatment options.
SCLC comprise about 20% of lung cancers and are the most aggressive and rapidly growing of all lung cancers. SCLC are strongly related to cigarette smoking, with only 1% of these tumors occurring in nonsmokers. SCLC metastasize rapidly to many sites within the body and are most often discovered after they have spread extensively. Referring to a specific cell appearance often seen when examining samples of SCLC under the microscope, these cancers are sometimes called oat cell carcinomas.
NSCLC are the most common lung cancers, accounting for about 80% of all lung cancers. NSCLC can be divided into three main types that are named based upon the type of cells found in the tumor: (i) Adenocarcinomas are the most commonly seen type of NSCLC in the U.S. and comprise up to 50% of NSCLC. While adenocarcinomas are associated with smoking, like other lung cancers, this type is observed as well in nonsmokers who develop lung cancer. Most adenocarcinomas arise in the outer, or peripheral, areas of the lungs. Bronchioloalveolar carcinoma is a subtype of adenocarcinoma that frequently develops at multiple sites in the lungs and spreads along the preexisting alveolar walls. (ii) Squamous cell carcinomas were formerly more common than adenocarcinomas; at present, they account for about 30% of NSCLC. Also known as epidermoid carcinomas, squamous cell cancers arise most frequently in the central chest area in the bronchi. (iii) Large cell carcinomas, sometimes referred to as undifferentiated carcinomas, are the least common type of NSCLC. (iv) Mixtures of different types of NSCLC is also seen.
Regardless of histopathologic subtype, the 5-year survival rate for lung cancer is 10-15%, which is the lowest among all cancers (Hoffman P C, et al. Lancet 2000; 355:479-85). This is mainly due to more than 60% of the patients diagnosed with advanced or metastatic disease, reflecting the need for a better understanding of the mechanisms that underlie lung carcinogenesis (Granville C A, et al. Am J Respir Cell Mol Biol 2005; 32:169-76.). Surgical treatment remains the main treatment modality for lung cancer, but it is possible only for people who are diagnosed at an early stage of cancer. Surgical candidates diagnosed with stage I NSCLC have 5-year relative survival rates of 52% (Reed M F, et al. Am J Surg 2004; 188:598-602) that is significantly better than the expensive chemotherapy to increase the median survival to be in the range of only two to four months. Therefore, the early diagnosis of lung cancer is critical for life span and successful therapy.
Typical diagnosis of lung cancer combines x-ray with sputum cytology. Unfortunately, by the time a patient seeks medical attention for their symptoms, the cancer is at such an advanced state it is usually incurable. Consequently, research has been focused on early detection of tumor markers before the cancer becomes clinically apparent and while the cancer is still localized and amenable to therapy.
Classical screening procedures, such as chest radiography and sputum cytology, have not decreased the mortality of lung cancer (Humphrey L L, et al. Ann Intern Med 2004; 140:740-53). Spiral computed tomography with multitrack scanners and autofluorescence bronchoscopy offers high sensitivity to detect lung cancer, even at the pre-invasive stage (Gohagan J K, et al. Lung Cancer 2005; 47:9-15, McWilliams A, et al. Curr Opin Pulm Med 2005; 11:272-7). However, low specificity and expensive cost could be serious issues with this method. Serum biomarkers have emerged as potential targets for progression monitoring of lung cancer, yet current biomarkers have not been adequately validated as an effective clinical tool for early screening and diagnosis (Sung H J, et al. BMB Rep 2008; 41:615-25). There is an emergent need for valid diagnostic procedures aimed at screening lung cancer at an early stage. Breath analysis, an easily and non-invasive technique, is one of the most desirable methods to identify new biomarkers for lung cancer.
Exhaled breath comprises volatile compounds and non-volatile compounds. The volatile compounds include gaseous molecules, such as carbon monoxide, nitric oxide, alkanes and benzene derivatives (Pauling L, et al. Proc Natl Acad Sci USA 1971; 68:2374-6, Gordon S M, et al. Clin Chem 1985; 31:1278-82). Volatile organic compounds (VOCs) of exhaled breath frequently served as analytes for clinical assay and several VOCs were identified as biomarkers for lung diseases (Kharitonov S A, et al. Chest 2006; 130:1541-6, Koutsokera A, et al. Curr Med Chem 2008; 15:620-30). In addition, VOCs were also applied to the diagnosis of lung cancer and to follow up the prognosis of tumor resection (Poli D, et al. Respir Res 2005; 6:71, Phillips M, et al. Cancer Biomark 2007; 3:95-109, Poli D, et al. Acta Biomed 2008; 79 Suppl 1:64-72.). The non-volatile compounds include small molecules, such as nitrites, nitrates and hydrogen peroxide, and larger molecules, such as eicosanoids, proteins, and DNA (Mutlu G M, et al. Am J Respir Crit Care Med 2001; 164:731-7, Montuschi P, et al. J Allergy Clin Immunol 2002; 109:615-20, Shahid S K, et al. Am J Respir Crit Care Med 2002; 165:1290-3.). Exhaled breath condensate (EBC) can be collected by guiding and cooling exhaled air in a condenser system. EBC has been used to measure and detect various inflammatory airway diseases, including bronchial asthma, cystic fibrosis and COPD (Carpagnano G E, et al. Chest 2004; 125:2005-10, Liu J, et al. Respiration 2007; 74:617-23, Robroeks C M, et al. Pediatr Allergy Immunol 2008; 19:652-9, Samitas K, et al. Respir Med 2009; 103:750-6.). Compared with other respiratory conditions, there are relatively small numbers of studies focused on lung cancer using EBC (Dalayeris E, et al. Lung Cancer 2009; 64:219-25, Chan H P, et al. Lung Cancer 2009; 63:164-8.). Therefore, this is a potential field for more studies to investigate the tumorigenesis processes and identify new biomarkers in the airway.
Identification of antigens associated with the lung cancer proteome has been of particular interest. These antigens have been used in screening, diagnosis, clinical management, and potential treatment of lung cancer. For example, carcinoembryonic antigen (CEA) has been used as a tumor marker of several cancers, including lung cancer. (Nutini, et al. 1990. Int J Biol Markers 5:198-202). Squamous cell carcinoma antigen (SCC) is another established serum marker. (Margolis, et al. 1994. Cancer 73:605-609.). Other serum antigens for lung cancer include antigens recognized by monoclonal antibodies (MAb) 5E8, 5C7, and 1F10, the combination of which distinguishes between patients with lung cancer from those without. (Schepart, et al. 1988. Am Rev Respir Dis 138:1434-8). Serum CA 125, initially described as an ovarian cancer-associated antigen, has been investigated for its use as a prognostic factor in lung cancer. (Diez, et al. 1994. Cancer 73:136876). Other tumor markers studied for utilization in multiple biomarker assays for lung cancer include carbohydrate antigen CA19-9, neuron specific enolase (NSE), tissue polypeptide antigen (TPA), alpha fetoprotein (AFP), HCG beta subunit, and LDH. (Mizushima, et al. 1990. Oncology 47:43-48; Lombardi, et al. 1990. Chest 97:639-644; and Buccheri, et al. 1986. Cancer 57:2389-2396).
Monoclonal antibodies to the antigens associated with lung cancer have been generated and examined as possible diagnostic and/or prognostic tools. For example, monoclonal antibodies for lung cancer were first developed to distinguish non-small cell lung carcinoma (NSCLC) which includes squamous, adenocarcinoma, and large cell carcinomas from small cell lung carcinomas (SCLC). (Mulshine, et al. 1983. J Immunol 121:497-502). Other antibodies have also been developed as immunocytochemical stains for sputum samples to predict the progression of lung cancer. (Tockman, et al. 1988. J Clin Oncol 6:1685-1693). U.S. Pat. No. 4,816,402 discloses a murine hybridoma monoclonal antibody for determining bronchopulmonary carcinomas and possibly adenocarcinomas. Some monoclonal antibodies utilized in immunohistochemical studies of lung carcinomas include MCA 44-3A6, L45, L20, SLC454, L6, and YH206. (Radosevich, et al. 1985. Cancer Res 45:5808-5812).
In U.S. Pat. Nos. 5,589,579 and 5,773,579, a lung cancer marker antigen specific for non-small cell lung carcinoma was identified and designated LCGA (also known as HCAVIII and HCAXII). U.S. Pat. No. 7,569,662 and U.S. Pat. App. Pub. No. 2009/0204334 disclose biomarkers for lung cancer. However, despite the numerous examples of isolated lung cancer antigens and subsequent production of MAb to these antigens, none has yet emerged that has changed clinical practice. Thus far, the immunoassays developed have failed to meet the need for early detection. Overall, despite the identification and extensive study of several potential tumor markers, none has been found to have clinical utility as a diagnostic marker or screening tool for lung cancer. It seems probable that given the complexity of the genetic and molecular alterations that occur in lung cancer cells, the expression pattern of these complex changes may hold more vital information in screening, diagnosis and prognosis than the individual molecular changes themselves.
Proteolysis-inducing factor/dermcidin (PIF/DCD) is a novel human gene, located on chromosome 12, locus 12q13.1, that encodes a secreted 110-amino acid protein. Two transcripts for the protein have been identified in normal skin, breast, placenta and brain, and in various primary and metastatic tumor cells. (Majczak, G., et al., Genet. Mol. Res. 6 (4):1000-1011 (2007)). DCD was originally identified as an antimicrobial peptide secreted by sweat glands (Schittek B, et al. Nat Immunol 2001; 2:1133-7). In addition to its antimicrobial function, DCD acts as a survival-promoting factor by means of enhancing cell growth during tumorigenesis in breast carcinoma (Porter D, et al. Proc Natl Acad Sci USA 2003; 100:10931-6) as well as in hepatic carcinoma (Lowrie A G, et al. Br J Cancer 2006; 94:1663-71) and prostate cancer cells (Stewart G D, et al. Prostate 2007; 67:1308-17). Moreover, PIF-CD is reported to induce skeletal muscle proteolysis, causing cancer cachexia (Monitto C L, et al. Clin Cancer Res 2004; 10:5862-9, Lee Motoyama J P, et al. Biochem Biophys Res Commun 2007; 357:828-33). Dermcidin acts as a survival factor in a variety of cancer cell lines under hypoxia or oxidative stress. (Stewart G D, et al. Br J Cancer. 2008; 99(1):126-32.) Dermcidin has been shown to be among secretory proteins that are up-regulated stage-specifically with stage IA or IIIA non-metastatic lung adenocarcinoma. (Nishimura T. et al., J Proteomics. 2009 Nov. 27. [Epub ahead of print]).
Recent technological advances in proteomics have permitted the development of diagnostic tests for the detection of some cancers. For example, one such technology includes the ProteinChip® surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) (Kuwata, H., et al., Biochem. Biophys. Res. Commun. 245:764-773 (1998); Merchant, M. et al., Electrophoresis 21:1164-1177 (2000)). This system uses surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry to detect proteins bound to a protein chip array. The SELDI system is an extremely sensitive and rapid method that analyzes complex mixtures of proteins and peptides. Applications of this technology show great potential for the early detection of prostate, breast, ovarian, bladder, and head and neck cancers (Li, J., et al., Clin. Chem. 48:1296-1304 (2002); Adam, B., et al., Cancer Res. 62:3609-3614 (2002); Cazares, L. H., et al., Clin. Cancer Res. 8:2541-2552 (2002); Petricoin, E. F., et al., Lancet 359:572-577 (2002); Petricoin, E. F. et al., J. Natl. Cancer Inst. 94:1576-1578 (2002); Vlahou, A., et al., Amer. J. Pathology 158:1491-1502 (2001); Wadsworth, J. T., et al., Arch. Otolaryngol. Head Neck Surg. 130:98-104 (2004)). For example, PCT Patent Application No. WO/2005/034727 describes the use of SELDI ProteinChip® technology as a tool of interrogation for head and neck squamous cell carcinoma (“HNSCC”) patients. This application describes how serum from HNSCC patients was compared to normal controls in order to develop HNSCC protein fingerprints for the diagnosis of HNSCC. However, to date, the use of SELDI had not been used to identify protein biomarkers for the detection of lung cancer.
Proteomic technologies have offered significant opportunities to discover clinical biomarkers (Kikuchi T, et al. Respirology 2007; 12:22-8). Mass spectrometry (MS) is one of the key tools of proteomic research to eliminate many of the limitations of traditional protein analyses. Also, tandem mass spectrometry (MS/MS) has become routine for peptides/proteins identification. The low-molecular-weight proteome, termed peptidome, provides a rich source of information for cancer diagnosis (Traub F, et al. Lab Invest. 2006; 86:246-53, Chang W C, et al. Proteomics Clin Appl 2008; 2:55-62.). Peptides, such as hormones, growth factors and cytokines, often possess specific functions in many physiological processes. The exploration of endogenous peptides, created by enzymatic cleavage of proteins in particular cellular environments, can result in relevant biomarker candidates (Petricoin E F, et al. Nat Rev Cancer 2006; 6:961-7, Schrader M, et al. Disease Markers 2006; 22:27-37). Coupling with low flow-rate capillary chromatography, the sensitivity of peptide detection by MS can reach attomole level (Amberoid R, et al. Nature 2003; 422:198-207), which holds great promise for biomolecular microanalysis. Furthermore, peptide sequence can be determined directly by MS/MS analysis without the need of sample manipulations, such as trypsin digestion. The identification of peptide marker is more convenient than conventional biomarker research.
Continued efforts to identify protein profiles or patterns that differentiate cancer from non-cancer could lead to earlier detection of lung cancer and the development of diagnostic tests for lung cancer. There is a need for methods and compositions for the diagnosis of lung cancer that are clinically useful.